Citrullus colocynthis (CC) is a natural herb plant that has traditionally been used in folk remediation. Anti-diabetic, antioxidant, and anti-inflammatory attributes are some of the CC medical properties. The present study aims to investigate the immune modulator potentials of CC seeds supplementation to chronically heat- stressed-exposed broilers. Results demonstrated that CC seeds supplementation to heat-stressed broilers was able to alleviate the negative impacts of heat stress on broiler immune responses and antioxidant status. Thus, CC seeds are suggested to be added to broilers reared under heat stress in order to improve the immune response and consequently ameliorate productivity.Chronic heat-stress exposure directly affects broiler immune response. Immunosuppression was demonstrated in broiler exposed to chronic heat stress with deterioration in humoral and cell-mediated immune responses. Natural immune modulator material is considered as a safe material for human consumption of poultry products. Citrullus colocynthis seeds (CCs) supplementation to modulate the immune response of broilers subjected to chronic heat stress. A total of 300 Cobb-500 male broiler chickens aged 21 days were randomly divided into two equal groups and reared under either thermo-neutral condition (24 ± 1 °C) or subjected to cyclic heat stress (34 ± 1 °C for 8 h). Each group was further divided into two groups (5 replicate × 15 chicks) and was fed either the basal diet or the basal diet with 0.1% CCs supplementation. The results showed that heat stress impaired the production performance by lowering the final body weight and feed intake as well as impairing feed conversion. The levels of stress markers  increased (p < 0.05), whereas the activity of antioxidant enzymes decreased in broilers exposed to heat stress. Further, heat stress caused direct suppression of broiler humoral and cell-mediated immune responses. The stimulating index of T and B lymphocytes proliferation, as well as the antibody titer against sheep red blood cells, were significantly (p < 0.05) reduced by heat-stress exposure. However, CCs supplementation to broilers subjected to heat stress improved (p < 0.05) the final body weight, feed intake, and feed conversion ratio (FCR), compared to the non-supplemented stressed group. The cellular and cell-mediated immune response indicators significantly enhanced (p < 0.05) with CCs supplementation. Supplementation of CCs to broilers reared under similar environmental conditions elevated the total white blood cells (TWBCs) count and the broiler stimulating index of T and B lymphocytes. It can be concluded that CC seeds can be effectively used to stimulate the immune response and improve the production performance of broilers reared under heat-stress condition.There is an extensive search for natural products that can be introduced to broiler rations to improve performance, especially during the unfavorable breeding conditions. Under heat-stress conditions, the immune response seriously deteriorates, which consequently impairs broiler production performance. Thus, the present study aimed to investigate the potentials of  In contemporary broiler industry, one of the challenging conflicts to maintain normal production performance is achieving optimum immune response. Heat-stress exposure mediates a number of physiological and behavioral changes that directly impair broiler production performance and cause a severe immunosuppression ,2,3. IrrCitrullus colocynthis (CC), bitter apple fruit, is an annual plant grown in the desert. C. colocynthis has traditionally been known to have pharmaceutical activities. Fruit pulp, seed, leaf, and root are altogether considered to have medicinal properties [operties . The medoperties ,18,19,20operties . Ten biooperties . Anti-inoperties ,24,25 asoperties . Normal operties  and withoperties . In a raoperties . HoweverAll experimental protocols were approved by the Research Ethics Committee (REC) at King Faisal University (KFU-REC/2021-02-17). To minimize birds suffering, heat-stress groups were monitored closely during the heat wave to detect any signs of chronic stress, such as breathing difficulty, watery discharge of the peak, decreased appetite, ruffled feathers, or droopy looking throughout the experimental period. Accordingly, if any of chronic stress sign appears, cervical dislocation was applied immediately to allow humane endpoints. Dry ripe CC fruits were obtained from different herbalists in Al-Ahsa, Saudi Arabia. Fruits were sliced and the seeds were manually isolated from the pulp. Mature black seeds were selected and then pulverized in a grinder . The resultant powder was collected and kept in dried containers at 4 °C until later use in the experiment.https://www.cobb-vantress.com/enUS/products/cobb500/; accessed on 15 January 2021). A brooding temperature was maintained at 33 °C for the first 3 days of age and decreased to 30 °C for the rest of the first week. Then, the temperature was gradually reduced (3 °C per week) to reach 24 °C at 21 days of age. The lighting program was set during the first 3 days of age at 24 L: 0D, and at 23 L: 1D for the rest of the experimental period. At 21 days of age, broiler chicks were symmetrically divided into 2 × 2 factorial design, each group contained 5 replicates of 15 broiler chickens. Each replicate of birds was reared in a floor pen (1.35 m lengths × 1.35 m widths) with 5-cm deep hardwood shavings litter. Starting from 22 to 42 days of age, broilers in the first chamber were exposed to 24 ± 1 °C and 50% RH representing thermo-neutral condition (control group), while broilers in the second chamber were exposed to cyclic heat stress as a heat wave from 9:00 a.m. to 5:00 p.m. at 34 ± 1 °C temperature and 50% RH representing chronic heat stress (HS group). Under the same environmental conditions, birds in each chamber were further divided into two equal groups. One of them was given the basal diet . The productive performance traits were recorded for each treatment group as described below. At the end of the experimental period (42 days of age), blood samples from birds of all treatment groups were taken and prepared for further assays of some stress indicators, antioxidant markers, immunological parameters, and some biochemical measurements.A total of 300 one-day-old male broiler chicks (Cobb500™) were purchased from a local hatchery and were raised in two identical environmental closed-system floor house chambers with similar conditions of size, ventilation, humidity, temperature, light intensity, and light schedule. Birds were allowed free access to feed and water that met NRC (1994) recommendations and the guidelines of Cobb500  was determined for each experimental group replicates considering each replicate pen as experimental unit. Feed intake  was determined for each treatment group by subtracting feed residual from the total offered amount. Then, feed conversion ratio was calculated  for each treatment group. The body weight, total feed offered and feed residual were determinate using digital electronic balance with 0.1 g sensitivity and 20 g accuracy/200 kg weight capacity .g, and the plasma was separated and kept at −20 °C for the analysis of corticosterone, and some biochemical measurements, such as total protein (TP), albumin, globulin, aspartate aminotransferase (AST), alanine transferase (ALT), creatinine, and urea.At the end of the experimental period (42 days of age), ten birds from each treatment group (2 birds per replicate) were randomly selected and blood samples (≈3 mL each) were withdrawn from the brachial vein using heparinized syringes. Small drops of the whole blood were used to evaluate some hematological parameters such as total white blood cells (TWBC) count and heterophils/lymphocytes (H/L) ratio. Samples were then centrifuged for 20 min at 4 °C and 1800× Another ten blood samples were obtained from each treatment group  for the isolation of peripheral blood mononuclear cells (PBMCs) according to the method previously described by Abass, et al. . PBMCs wFurther blood samples (approximately 3 mL) were collected at the end of the experimental period from ten birds in each treatment group (2 birds per replicate). PBMCs were obtained from these samples and processed freshly to measure the proliferation activity of T- and B-lymphocytes.n = 10; 2 samples per replicate) were prepared for each treatment group, and leukocytes were stained using Hema-3 stains . The differential leukocyte counts were performed for a total of 200 leukocytes in two different slides using a light microscope (a magnification of 1000× with oil immersion) and the H/L ratio was then calculated.H/L ratio was determined according to the methodology described by Mehaisen, et al. . Blood sn = 10; 2 samples per replicate in each group) using a specific chicken corticosterone ELISA kit . The intra- and inter-assay coefficient of variations (CV) were <8% and 10%, respectively. The assay sensitivity was less than 0.0625 ng/mL, with a dynamic range of 0.5 to 20 ng/mL. The measurements were recorded by an automated microplate reader .Plasma corticosterone concentrations were determined  were quantified using ELISA kits specifically designed for chicken , and the measurements were recorded by the automated microplate reader. According to the manufacturer, the intra-assay and inter-assay CV were less than 8% and 12% for the MDA, and 5.57% and 5.89% for the TNFα, respectively. The detection ranges were 1.56–100 ng/mL and 31.25–2000 pg/mL for MDA and TNF-α, respectively.The collected and stored PMBCs pellets were re-suspended in 1 mL PBS, kept on ice for 1 min, and then sonicated for another 1 min. The homogenized cells were centrifuged at 1030× n = 10; 2 samples per replicate in each group). As mentioned above, the PBMCs pellets were homogenized, centrifuged, and the supernatant was used for the assay. The levels of TAC and SOD were determined in the supernatant following the instructions of colorimetric assay kits , and the measurements were obtained using the automated microplate reader. The average intra- and inter-assay CV were 2.7% and 8.2% with a detection range of 0.62–145.2 U/mL for TAC, while the intra- and inter-assay CV were 5.1% and 9.6% with a detection range of 1.35–62 U/mL for SOD.TAC and SOD were determined as antioxidant markers in the PBMCs  was analyzed by Western blot technique using the protocol described by Abass, Kamel, Khalifa, Gouda, El-Manylawi, Mehaisen and Mashaly [β-actin antibody  was added and incubated to the membrane to confirm equal loading of samples followed by a horse radish peroxidase conjugated goat anti-mouse IgG . Detection of HSP-70 was then performed using the ECL chemiluminescence kit .The expression of HSP-70 in the PBMCs pellets  was performed according to the methods previously described by Gehad, et al. [TWBCs count  into the toe web of the left foot between the third and the fourth digit. The swelling response to the mitogen injection was calculated as the difference in mm between the thickness of the toe web measured immediately before PHA-P injection and after 24 h of injection. Broiler cell-mediated immune response was assessed using the magnitude of toe web swelling induced by intradermal phytohemagglutinin (PHA-P) mitogen injection as described in a previous work . Brieflyg. The anti-SRBCs AB titers were quantified by using the micro-hemagglutination technique, and the titer was expressed as log2 of the reciprocal of the highest dilution giving complete agglutination [Ten broiler chicks from each experimental group (2 chicks per replicate) were assigned to evaluate the anti-SRBCs AB as a measurement of broiler humoral-mediated immune response. At 42 days of age, birds were intravenously injected with 1 mL of saline suspension of 5% SRBCs. Blood samples were collected one week later, then sera were separated by centrifugation at room temperature, 220× tination .n = 10; 2 samples per replicate in each group) were detected from washed PBMCs using trypan blue dye . The viable cells were then plated in 96-well plate at 6 × 106 cells per well. To stimulate and estimate T- and B-lymphocytes proliferation, 50 mL of either concanavalin-A  or lipopolysaccharide  was used; whereas, 50 mL of RPMI-1640 medium  was added to the un-stimulated control cells. Cells were then incubated for 48 h at 42 °C and 5% CO2. Afterwards, 15 mL of 3--2,5-diphenyl-2H-tetrazolium bromide (MTT)  was added to each well, then cells were incubated for 4 h at 42 °C. After incubation, 100 μL of 10% sodium dodecyl sulfate, dissolved in 0.04 M HCl, was added into each well. The absorbance was detected using ELISA microplate reader at 570 nm. Stimulating indexes (SI) of T- and B-lymphocytes proliferation were then calculated as the ratio of stimulated cells optical density against the un-stimulated control cells optical density.The stimulating index (SI) of T- and B-lymphocyte proliferation was determined according to the methods described in a previous work Abbas, et al. . Brieflyn = 10; 2 samples per replicate in each group) were performed following the manufacturer instructions of available colorimetric assay kits and data were obtained using an automated microplate reader. The plasma TP, albumin (A), ALT, and AST concentrations were determined following the instructions of the kit manufacturers  as indicators for the broiler liver functions. The globulin concentration (G = TP–A) and A/G ratio were then calculated for each treatment group. Meanwhile, the plasma creatinine and urea concentrations were measured by using commercial kits  to evaluate the broiler kidney function. The plasma biochemical analyses . Heat stress, CC seeds supplementation and their interaction were set as fixed effects. Post hoc analysis was performed to compare means using Duncan’s test and the significance was set at p < 0.05. Results were expressed as mean ± SEM.Data were subjected to the analysis of variance using the GLM of SASCitrullus colocynthis seeds (CCs) supplementation is presented in p < 0.05) in the final body weight, body weight gain (BWG), and feed intake by 28, 41, and 24%, respectively, compared to the thermo-neutral control group. Feed conversion ratio (FCR) also showed higher values (p < 0.05) for broilers exposed to heat stress, reflecting poor productivity. However, CCs supplementation significantly (p < 0.05) enhanced FCR compared to non-supplemented broiler chickens that received basal diet. In addition, under heat-stress condition, broiler chickens supplemented with CCs showed better (p < 0.05) FCR compared to non-supplemented stressed group. Nevertheless, the statistical analysis did not confirm any significant differences in the other production parameters between CCs supplemented and non-supplemented groups reared under the thermo-neutral control conditions. However, under the heat-stress conditions, CCs-supplemented-stressed group was able to increase (p < 0.05) the final body weight and BWG by 14 and 23%, respectively, with a significant improvement in FCR in comparison with the non-supplemented stressed group.Broiler production performance under thermo-neutral or heat-stress conditions and with or without p < 0.05) by 4.1-folds in the heat-stressed group compared to the thermo-neutral control group. Moreover, an exponential increase in TNF-α and MDA levels of 2- and 2.8-folds, respectively, was observed in the heat-stressed group compared to the thermo-neutral control group. Furthermore, the H/L ratio was increased by 2.4-folds in the heat-stressed group, indicating long-term stress exposure. These findings confirm the induction of oxidative stress as a consequence of heat-stress exposure. However, there was a significant (p < 0.05) positive effect of CCs supplementation to the heat-stressed broilers with a reduction in the stress indicators and an increase in the antioxidant markers when compared to the non-supplemented stressed group.Results showed that stress indicators  were significantly increased, whereas the SOD activity and TAC were decreased when the broilers were exposed to heat-stress condition . From onThe HSP-70 Western blot output of broilers subjected to either thermo-neutral or heat-stress conditions and with or without CCs supplementation is presented in p < 0.05) immunosuppression effect of heat stress on both humoral and cellular levels. The TWBCs count were deteriorated (p < 0.05) with heat-stress exposure. Further, a reduction (p < 0.05) in the humoral  as well as cell-mediated (toe web swelling) immune response was associated with heat-stress exposure. However, the supplementation of CCs to the heat-stressed broilers demonstrated positive impacts on all the examined immunological parameters. Moreover, CCs supplementation was able to bring TWBCs count as well as T- and B lymphocytes stimulating index and the antibody titer against SRBCs to the normal values of the thermo-neutral control group. Interestingly, under the same environmental conditions, CCs supplementation significantly (p < 0.05) increased the TWBCs count and boosted the T- and B-lymphocytes proliferation index. To assess dietary CCs immune-modulation potentials under thermo-neutral and heat-stress conditions, different humoral and cell-mediated immune assays were performed . The resp < 0.05) increased the plasma total protein associated with a reduction in the albumin concentration and A/G ratio level, and an increase in the globulin concentration. Furthermore, the activity ALT and AST as well as the levels of creatinine and urea were significantly increased, indicating negative impacts of heat stress on both liver and kidney functions. Remarkably, under heat-stress conditions, CCs supplementation significantly alleviated the negative impact of heat stress on the liver enzymes activity and kidney function markers as well as the plasma albumin level. Meanwhile, the interaction effect of heat stress and CCs supplementation was significant on all the measured blood biochemical parameters except for plasma A/G ratio, plasma ALT activity, and plasma urea concentration. It is crucial to note that CCs supplementation, under thermo-neutral condition, did not show any adverse toxic effects on either broiler’s liver or kidney functions. Plasma biochemical parameters of broilers reared under thermo-neutral or heat-stress condition with or without CCs supplementation are presented in The current experiment illustrated the negative impact of heat stress on broiler production performance and immune response. The immunosuppression was confirmed with the deterioration in both cellular and humoral immune responses resultant of heat-stress exposure. The negative correlation between heat-stress exposure and immune response was demonstrated earlier . One of Heat stress is known to induce oxidative stress and excessive ROS production . BroilerA wide range of environmental or metabolic stress exposure induces over-expression of HSPs in order to protect cells from damage ,50,51. IBlood biochemical profile reflects the physiological fitness of birds. In the present study, a significant negative effect of heat stress was observed in both liver and kidney functions. The reduction in albumin levels and the increase in creatinine, urea, and uric acid levels, as well as the increase in AST and ALT activities were associated with heat-stress exposure ,14,45. LIt can be inferred from the present study that heat stress induces immunosuppression by the excessive release of corticosterone and pro-inflammatory cytokines. Subsequently, the proliferation and differentiation of cellular and humoral immune cells were significantly reduced. Such unbalanced immune response directly influenced the production performance. However, CC seeds supplementation to heat-stressed broiler chickens was able to interrupt the cascade of inflammation-immunosuppression pathway. This interruption reduced the corticosterone and pro-inflammatory cytokine production, which subsequently alleviated the cellular and humoral immunosuppression. Thus, CC seeds can be supplemented to heat-stressed broilers to alleviate the negative impact of heat stress on production performance and regain immune response homeostasis. Further studies are needed to confirm the immune stimulation effect of CC seeds when applied at different levels.


Local chest irradiation decreased the level of l-Histidine in fecal pellets, which was increased following fecal microbiota transplantation. l-Histidine replenishment via an oral route retarded the pathological process of lung and heart tissues and improved lung respiratory and heart systolic function following radiation exposure. l-Histidine preserved the gut bacterial taxonomic proportions shifted by total chest irradiation but failed to perform radioprotection in gut microbiota-deleted mice. ImP, the downstream metabolite of l-Histidine, accumulated in peripheral blood and lung tissues following l-Histidine replenishment and protected against radiation-induced lung and heart toxicity. Orally gavaged ImP could not enter into the circulatory system in mice through an antibiotic cocktail treatment. Importantly, ImP inhibited pyroptosis to nudge lung cell proliferation after radiation challenge. Together, our findings pave a novel method of protection against cardiopulmonary complications intertwined with radiotherapy in pre-clinical settings and underpin the idea that gut microbiota-produced l-Histidine and ImP are promising radioprotective agents.Radiation-induced cardiopulmonary injuries are the most common and intractable side effects that are entwined with radiotherapy for thorax cancers. However, the therapeutic options for such complications have yielded disappointing results in clinical applications. Here, we reported that gut microbiota-derived  Cancer ranks as the top public enemy and is the illness with the highest incidence and largest patient number in the world. Because of technological advancements, the number of cancer survivors is gradually reaching 33 million . In deveThe gut microbiome plays important roles in human health . Recentll-Histidine/ImP axis lulled the pathological processes of the lung and heart tissues and ameliorated lung respiratory and heart systolic functions following radiation stimuli in a gut microbiota-dependent fashion. Mechanistically, ImP blocked pyroptosis to propel the proliferation of pneumonocytes. Together, our findings provide novel insights into the function and the underlying protective mechanism of microbiota metabolites in the context of radiation-induced cardiopneumatic toxicity in preclinical settings.In this study, we aimed to screen bioactive compounds from gut microbiota-derived metabolites to battle against radiation-induced cardiopneumatic injuries. Our observations demonstrated that the l-Histidine in fecal pellets, which was reversed following fecal microbiota transplantation  value, decreased VO2 intake emissions, and alleviated pneumonia, as judged by the reduction of radiation-elevated pro-fibrotic and pro-inflammatory factors  decreased the level of ion FMT, . Given tl gavage . Next, S process C,D. l-Hiwing TCI E, sugges factors . In line tissues I,J, reco tissues , and les tissues . Togethel-Histidine in the fecal pellets, 16S rRNA sequencing was performed to analyze whether l-Histidine impacted gut microbiota with radiation stimuli. Local chest irradiation heightened the α-diversity of the intestinal bacteria, while l-Histidine erased the elevation  was added to the drinking water for one of the two groups. As shown by the Sirius Red and Masson staining, gut microbiota deletion interfered with l-Histidine-protected induced structural damage and collagen accumulation in local chest-irradiated mice phenyl 3-chlorobenzoate and abscisic acid but decreased that of l-aspartic acid  following radiation challenge, indicating that ImP, a secondary metabolite of l-Histidine, is able to fight against radiation-induced pneumonic injury  in a dose-dependent manner after radiation exposure; however, ImP represented more obvious radioprotection  has been reported to protect against radiation pneumonia . Howeverne daily . l-Histi failure . l-Histi surgery . In addi surgery  and can  surgery . In the l-Histidine can be catabolized into two classic secondary metabolites, imidazole propionate and histamine. Therefore, it is interesting to unravel whether l-Histidine protects against irradiation directly or relies on the downstream metabolites. To answer the question, we performed a battery of experiments. First, we used ABX to clean gut microbes. The gut microbiota-deleted mice did not respond to l-Histidine treatment and exhibited similar radiation-induced cardiopulmonary injuries to the chest of the local-irradiated controls. This suggests that destructing the metabolic function of the gut flora lull the radioprotective effects of l-Histidine. Secondly, we assessed the levels of ImP and histamine in PB and lung tissues from experimental mice with l-Histidine replenishment. The results show that only ImP was enriched in the target tissues. In addition, the oral gavage of ImP ameliorated radiation-induced cardiopulmonary toxicity, indicating that ImP might be the key secondary metabolites of l-Histidine to fight against radiation injuries. Finally, the experimental mice were treated with vancomycin or streptomycin, respectively. Results showed that the oral gavage of ImP cannot be enriched in PB and lung tissues in vancomycin-treated, Gram-positive bacteria-deleted mice, suggesting that gut commensal Gram-positive bacteria might play key roles in the absorption of ImP. Together, all of the evidence suggests that the gut microbiota might catabolize l-Histidine into ImP and assist in the entry of ImP into the circulatory system to protect against radiation-induced cardiopulmonary toxicity.l-Histidine, activated different signaling pathways to perform radioprotective functions in radiation-induced lung injury. Thus, our findings indicate that ImP fight against radiation-induced cardiopulmonary injuries through inhibiting pyroptosis.Acute pneumonia occurs within a few weeks after radiation and is characterized by the release of pro-inflammatory factors and the accumulation of immune cells in lung tissues. Research has reported that ionizing radiation precipitates pyroptosis in multiple tissues and organs  . PyroptoSix- to eight-week-old-male (around 20 g) C57BL/6J mice were purchased from the Beijing Huafukang Bioscience Co., Inc. . Mice were housed in the Specific Pathogen Free (SPF) level animal facility at the Institute of Radiation Medicine (IRM), the Chinese Academy of Medical Sciences (CAMS). All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at CAMS.2 and 37 °C.BEAS-2B cells were obtained from the American Type Culture Collection (ATCC) and were certified to be mycoplasma-free. The passage numbers of those cell lines during the experimental period were no more than eight. The cells were cultured with 10% fetal bovine serum , 100 U/mL penicillin, and 100 mg/mL streptomycin and were grown at 5% COl-Histidine  and imidazole propionate  at a dose of around 200 μL per mouse. Mice were sacrificed, and tissue samples were collected after the 21-day course of the experiment. BEAS-2B cells were exposed to 4 Gy (for colony formation assays) or 6 Gy (for other cell experiments).A Gammacell−40 137Cs irradiator  at a dose rate of 0.8 Gy per minute was used for all experiments. Mice treated with total chest irradiation (TCI) were exposed to 15 Gy γ-ray, and control mice were sham-irradiated. Mice were intragastrically administered g rpm for 3 min. The supernatant was obtained for treatment. Mice in the FMT group were gavaged with the supernatant 200 μL for 7 days before irradiation and then for 10 days after irradiation. TCI groups were given equal doses of placebo. The schematic diagram of FMT procedure is shown in the For FMT administration, donor stool was freshly prepared on the day of transplant and within 4 h of transplantation. Donor stool was weighed and diluted with 2 mL of saline per 0.1 g of stool. In brief, the stool was steeped in saline for about 15 min, shaken, and then centrifuged at 800× l-Histidine/ImP) group. Mice in the ABX group were housed with ABX fluid in their drinking water for 7 days before irradiation and then for 21 days after irradiation. The schematic diagram of the ABX procedure is shown in the ABX consisted of ciprofloxacin (0.125 g/L), metronidazole (0.1 g/L), vancomycin (0.05 g/L), streptomycin (100 U/L), and penicillin (100 U/L). Mice were exposed to ABX in drinking water after TCI. The same water was used for the water-treated (TCI + g and 4 °C. Serum was collected using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Serum was centrifuged for 15 min at 1000× g and 4 °C. Protein level was measured from the clear supernatant using an ELISA kit  according to the manufacturer’s protocols. The supernatant was obtained to assess the level of ImP using the ELISA kit  according to the manufacturer’s protocols . Optical density was read at 450 nm .  The l-Histidine (or propionate imidazole) level in the lung tissues and serum were assessed in l-Histidine (or propionate imidazole) replenishment mouse models  and ABX-treated mouse models (gut microbiota-deleted mice).Frozen lung samples were ground up and reconstituted in PBS to a final concentration of 0.1 g/300 μL, followed by centrifugation for 10 min at 8000× 2O2 was added to the slide specimens. The slide was put into a 0.01 M citrate buffer (pH 6.0) pot and was heated at 95 °C for antigen retrieval to be performed. After being blocked with normal goat serum, the prepared primary antibody should continue to be dropped and incubated overnight in a refrigerator at 4 °C. On the second day, the biotin-labeled goat anti-rabbit secondary antibody working solution was incubated at room temperature for 10 min; the HRP-labeled horseradish peroxidase-labeled working solution was incubated at 37 °C for 10 min; the slices were dripped with newly configured DAB chromogenic solution; hematoxylin was counter-stained for 2 min; finally, the slides were dehydrated and mounted with neutral gum.First, the slides were baked at 65 °C, and then the xylene solution was dewaxed, and different gradients of alcohol were dehydrated. After the dewaxing was completed, 3% HFor colony formation analysis, 48 h after irradiation, 500 viable irradiated (transfected) cells were placed in 6-well plates and were maintained in complete medium for 2 weeks. Then, colonies were fixed with methanol and were stained with methylene blue.g for 10 min at 4 °C.Mice were sacrificed, and one side of the bronchus was ligated, and the airway on the other side was lavaged three times with 800 μL ice-cold PBS. A total of 80% of the input volume was recovered, which was defined as BALF. BALF was centrifuged at 500× 2/CO2), oxygen intake VO2 (mL/min/kg^0.75), and carbon dioxide emissions (VCO2 (mL/min/kg^0.75) of the mice in each group. Before monitoring, the system needed to be calibrated: low O2 (20%), high O2 (50%), low CO2 (0%), high CO2 (1.5%). Then, each mouse was weighed, and the average value was calculated. The mice were then placed in a metabolic cage. At this stage, the software should be opened in order to continuously monitor mice for 24 h, and the software should be used to calculate the relevant data.We used the Panlab Oxylet system  to monitor the 24-h RQ . This method uses a specialized volume pressure recording (VPR) sensor that are placed over the animal’s tail to measure blood volume changes. The mice do need to be restrained and artificially heated in specific holders in order to maintain normal BP.n) in each group. Significance was assessed by comparing the mean values using Student’s t-test and the Wilcoxon rank sum test for independent groups as follows: * p < 0.05; ** p < 0.01; *** p < 0.001.Each experiment was repeated at least three times. The normal distribution of the data was assessed using the Kolmogorov–Smirnov test. The data are presented as the means ± SD with respect to the number of samples (l-Histidine/ImP can protect against radiation-induced cardiopulmonary toxicity. Mechanistic investigation showed that the radioprotective function of l-Histidine or ImP is dependent of gut microbiota. Clinically, l-Histidine or ImP might be employed as a potential and promising radioprotective agent to fight against radiotherapy intertwined cardiopulmonary injury.Together, our study provides novel insights into the gut microbiota-derived metabolites and underpins that gut flora-produced


Mental health issue among college students is routinely a major public health concern, and coronavirus disease 2019 (COVID-19) pandemic may have exacerbated the students' mental health issues which include psychological distress, panic disorder, insomnia, and posttraumatic stress symptoms. However, few studies reached a consensus on the impact of COVID-19 fear on mental health among college students. Therefore, we aimed to conduct a systematic review and meta-analysis that quantitatively synthesized the fear among college students during the COVID-19 pandemic.PubMed, EMBASE, CINAHL, and PsycINFO electronic databases were systematically searched to identify cross-sectional study reporting the state of COVID-19 fear examined by the Fear of COVID-19 Scale (FCV-19S) published up until November 20, 2021. Methodological quality was complied with the evaluation criteria of the Agency for Healthcare Research and Quality. The random effects model was employed to estimate the pooled mean of FCV-19S score. Subgroup analysis and meta-regression analysis were also conducted. Publication bias was assessed by Begg's test and funnel plot.A total of 16 studies with a sample size of 11,872 were included. A pooled mean of FCV-19S score was 17.60 [95% confidence interval (CI): 16.41–18.78]. The mean of COVID-19 fear in women  was higher than that in men . The highest and lowest pooled means of FCV-19S score were observed in the studies conducted in multiple countries that include Israel, Russian, and Belarus  and in Europe , respectively. No significant publication bias was detected by Begg's test.College students experienced a moderate level of fear caused by COVID-19 pandemic. It is necessary to design and implement prevention programs that target the mental health of college students.https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021287017, identifier: CRD42021287017. Globally, the coronavirus disease 2019 (COVID-19) pandemic has led to over 249 million confirmed cases and more than 5 million deaths as of November 20, 2021 . Due to Fear is defined as an unpleasant emotional state that is elicited by the perception of danger and insecurity feelings . Fear, aCompared to the general population, college students are reported to be more vulnerable to sudden changes in COVID-19 pandemic . Studenthttps://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021287017). In this systematic review and meta-analysis, we aimed to estimate the pooled mean of fear of COVID-19 among college students.This systematic review and meta-analysis were conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines  Supplem, and theWe systematically searched PubMed (2019–2021), EMBASE (2019–2021), CINAHL (2019–2021), and PsycINFO (2019–2021) electronic databases to identify such literatures published up until November 20, 2021. The search terms were “2019 novel coronavirus-infected pneumonia” or “2019 novel coronavirus” or “2019 novel coronavirus pneumonia” or “COVID-19 pneumonia” or “COVID-19” or “2019-nCOV,” “undergraduate” or “university student” or “college student” or “higher education student,” and “anxiety” or “worry” or “fear” or “concern.” The detailed search strategy is shown in The inclusion criteria were as follows: (1) cross-sectional study; (2) the state of fear of COVID-19 was examined by the FCV-19S; (3) the mean and standard deviation of FCV-19S score were reported; (4) the study was conducted among college students; and (5) publications published in English. Studies were excluded based on the following criteria: (1) studies without sufficient data; (2) publication of review, conference summary, and other non-original research; (3) multiple participants that did not present results separately for college students; and (4) methodological quality score (<4).Two independent investigators (FW and LD) screened the articles and selected those having the search terms in their titles or abstracts. Full-text articles which initially met the selection criteria were included. The kappa coefficient was calculated for measuring the agreement between two investigators making inclusion or exclusion decision. Disagreements between two investigators were resolved through negotiation with a third researcher (LZ). Then, they extracted the following data using a predesigned electronic form. The following data included the name of first author, publication year, age, gender, sample size, country of participants, and mean and standard deviation of FCV-19S score. According to the evaluation criteria for an observational study of the Agency for Healthcare Research and Quality (AHRQ), the quality assessment of the included studies was performed by two independent investigators (FW and LD) and assigned the methodological quality score. AHRQ contains 11 items, and each item is answered as yes, no, or not reported. The score of the answer “yes” is 1, whereas “no” or “not reported” scores 0. In terms of methodological quality score, studies were defined as low (0 to 3), moderate (4 to 7), and high qualities (8 to 11) . Thus, wQ test and I2 statistics to detect the heterogeneity among eligible studies. A fixed effects model was applied to generate the pooled raw mean when few evidence of heterogeneity was found among these studies (I2 ≤ 50% and p for heterogeneity ≥ 0.10); otherwise, the random effects model was used. The forest plot was adopted to visually depict the heterogeneity among the studies and show the pooled raw mean of FCV-19S score of selected studies. Subgroup analyses by continent (Asia/Europe/America/Multiple), publication year, and gender were conducted to examine the source of heterogeneity. Meta-regression method was employed to evaluate the association of raw mean of FCV-19S score with mean age and sample size of participants in included studies. Leave-one-out sensitivity analysis by dropping out one study at a time to estimate the pooled raw mean of remaining studies was conducted to determine whether a single study was a significant source of heterogeneity. Publication bias was assessed by Begg's test and funnel plot. The statistical analyses were performed using R software version 4.1.1 with meta and metaphor packages, and all statistical tests were two-tailed. A p-value < 0.05 was considered statistically significant except for the examination of heterogeneity.Meta-analysis was employed to estimate the pooled raw mean of FCV-19S score and the corresponding 95% confidence interval (CI). We used Cochran's n = 64), study using other scales (n = 53), and letters–editorials–reviews (n = 76). In addition, 21 articles were eligible for the full-text screening, and 5 articles were excluded for insufficient data. Agreement between two independent investigators regarding the study selection was considered perfect (kappa coefficient = 0.79). Finally, 16 articles were included in the meta-analysis.The flowchart of literature search and study selection based on the PRISMA guidelines is shown in As shown in Q = 1,205.27, df = 15, p < 0.01, τ2 = 5.7737, I2 = 98.8%). The pooled raw mean of FCV-19S score was 17.60 (95% CI: 16.41–18.78)  , which s1–18.78) .p-value of 0.086  and in Europe , respectively . No signof 0.086 . Moreoveof 0.086 .p = 0.992; p = 0.738)  .p = 0.910) (As shown in = 0.910) .The present systematic review and meta-analysis included 16 studies with 11,872 participants. Our results showed that the pooled mean of FCV-19S score was 17.60 (95% CI: 16.41–18.78), which was lower than those in the general population and medical staff reported in a study by Luo et al. . Fear isThe pooled mean of FCV-19S score was higher in women than in men, which indicates that women experienced more fear during COVID-19 pandemic than men. This finding is consistent with the studies performed among the general population in Cuba and Bangladesh  and amonThis study found that the highest and lowest pooled means were observed in the items 1 and 3 of FCV-19S, respectively. This is likely attributed to what the items evaluate. Item 1 (I am most afraid of coronavirus 19) mainly assesses the attitude toward COVID-19, whereas item 3 (my hands become clammy when I think about coronavirus 19) especially refers to the hand symptoms of fear related to COVID-19. Similarly, participants received a score at the low level in item 6 (I cannot sleep because I am worrying about getting coronavirus 19). Both items 3 and 6 ask about symptoms of fear caused by COVID-19. Facing the spread of the COVID-19, a large number of people feel fear and anxiety, but only a small number of severely anxious individuals will have obvious symptoms that include sleepless and muscular tensing .Since the first case of COVID-19 was reported in Wuhan, China in late 2019, the pandemic has left the world lasting scars in both disastrous mortality and public panic. The results of this study indicated that there was no significant difference in the FCV-19S scores between studies published in 2020 and 2021. Furthermore, the emergence of SARS-CoV-2 variants has triggered new waves in many countries , 60. It Moreover, different prevalence of fear of COVID-19 among different countries can be attributed to cultural contexts and other contextual factors and differences in access to medical care and mental healthcare . For insSeveral limitations should be acknowledged in this study. First, studies included in the meta-analysis were cross-sectional studies, which reported the prevailing situation. Longitudinal studies are needed to examine the causal relationship between the mental health consequences and COVID-19 fear. Second, the FCV-19S score is not a clinically relevant value to be targeted by an intervention. However, the results of this study showed that college students experienced a moderate level of fear related to COVID-19. Third, only articles published in English were included in this meta-analysis, and qualified articles published in other languages were not included, which might affect the results.In conclusion, the results of the present systematic review and meta-analysis showed that college students have experienced a moderate level of fear caused by COVID-19, with female students having higher level of fear than male ones. Disparity in the prevalence of fear of COVID-19 among countries might be attributed to cultural contexts and access to healthcare services. It is necessary for educational authorities and universities to develop appropriate mental health interventions for the fear of COVID-19 among college students.The original contributions presented in the study are included in the article/YD and FW designed the study protocol. FW, LZ, and LD conducted the literature search. FW, LZ, and YD retrieved and selected the articles. FW, LZ, and LW conducted data extraction. FW and LZ performed the statistical analysis of the data. FW and YD wrote the manuscript draft. YD and LZ supervised the study. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of Shandong Province (Grant No. ZR2017PH012). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background and purpose — There is lack of knowledge concerning patient-reported long-time outcome after arthroplasty. Therefore, we investigated patient self-reported physical capabilities (PC) and subjective well-being (SW) up to 20 years after total hip (THA) or knee (TKA) arthroplasty.Subjects and methods — The self-reports from postal questionnaires for study checkpoints  were provided by the Kuopio OSTPRE study including only women aged 52–62 years . The Finnish Arthroplasty Register and Care Register for Health Care provided data on arthroplasties in the OSTPRE population. The results of women with THA/TKA were compared with women without arthroplasty (control group).Results — In subjects with THA performed before the 10-year follow-up, the proportion of good PC was initially decreased by 0.6 percentage points (pp) at the 10-year follow-up and later by 19 pp at the 20-year follow-up. After TKA, the proportion of subjects with good PC decreased by 4.1 pp (10–year follow-up) and 27 pp (20-year follow-up), respectively. The proportion of controls reporting good PC decreased by 1.4 pp at the 10-year follow-up and 14 pp at the 20-year follow-up compared with the baseline. After THA, the proportion of subjects with good SW stayed on the same level at 10-year follow-up and decreased by 2.3 pp at 20-year follow-up. After TKA, the proportion of good SW increased by 9.0 pp (10-year follow-up) and decreased by 14 pp (20-year follow-up). The proportion of controls reporting good SW increased by 4.0 pp (10-year follow-up) and decreased by 8.8 pp (20-year follow-up).Interpretation — THA and TKA maintain PC and SW. The overall PC and SW are lower in women with arthroplasty, in comparison with controls without arthroplasty. THA seems to outperform TKA in maintaining PC. In recent years, more attention has focused on patient-reported outcomes after total hip (THA) and knee (TKA) arthroplasty. Most studies on patient-reported outcome measures (PROM) have relatively short follow-ups  and subjective well-being (SW) in women even up to 20 years after a primary THA or TKA. We compare THA/TKA patients with a control group and postoperative scores were compared with preoperative scores.Supplementary data on all THAs and TKAs in the OSTPRE study population was obtained from the Finnish Arthroplasty Register (FAR) and the Care Register for Health Care (CRHC).This study is based on the long-term follow-up of the female population in the Kuopio Osteoporosis Risk Factors and Prevention study (OSTPRE). The self-reports on participants’ PC and SW were provided by OSTPRE. http://www.uef.fi/en/web/kmru/ostpre). The original study cohort included all 47–56-year-old women  living in Kuopio Province in Eastern Finland in 1989. The study is based on self-reports via postal questionnaires, and it has been renewed every 5 years. In the current study, the OSTPRE 1994 questionnaire  is used as baseline. Follow-ups are the 2004 , and 2014  questionnaires. We chose these questionnaires to achieve long enough follow-up times for the participants. We focused on questions concerning self-reported PC and SW. These questions have basically remained the same since the questionnaire in 1994. Only those who had returned all 3 questionnaires were included in the study. The self-reported hip fractures in OSTPRE, also included in the present study, were complemented with the hip fractures found from the CRHC and all were also checked from the medical records.The original purpose of the OSTPRE study was to investigate osteoporosis in the female population in a prospective study setting. However, it has expanded from its start in 1989 into an overall health and subjective well-being cohort, still including only a female population : “Describe your current physical capability?” and “How would you describe your current well-being?”. Self-reported original PC included the following answer options: 1, capable of moving without limitations; 2, no running, without other limitations; 3, can move less than 1,000 meters; 4, can move less than 100 meters independently; 5, can move only indoors; 6, I’m temporarily immobilized; 7, I’m permanently immobilized. For statistical purposes (group size), answers 1 and 2 were combined as the group “walking without limitations” and are referred later as “good PC.” Also, answers 4–7 were considered as one group, “can move less than 100 meters independently.” This classification works well in clinical settings too, since being able to walk less than 1,000 meters supports the indication for arthroplasty. Originally, SW answers formed 5 groups: very good, good, moderate, poor, and bad. Again, for statistical purposes, very good and good were combined as “good.” Poor and bad were combined as “poor.”THA/TKA register data was collected from the FAR and CRHC. We used 2 different data sources, since it has been previously found to more comprehensively cover all arthroplasties  to compare means of, e.g., height, weight, and BMI. We used propensity score matching to select the most suitable controls for women operated in with THA or TKA . The variables found in Characteristics  were useThe Research Ethics Committee of the Northern Savo Hospital District has given permission for the OSTPRE study (3/11/2014//78/2004). Written consent has been provided by every study participant. The Finnish Institution for Health and Welfare has granted permission to use the CRHC and FAR data (THL/20/5.05.00/2016). This study was supported by the Finnish Arthroplasty Association, Päivikki and Sakari Sohlberg Foundation and Academy of Finland. The authors have no conflicting interests to report.The overall study population consisted of 6,462 women, 292 of whom had THA and 527 whom had TKA. Hip fracture was the indication for THA in 9 women.Supplementary data). Both THA and TKA women reported maintained or improved good SW after operation, at the 10-year follow-up 33% (THA)/41% (TKA). Later, at the 20-year follow-up, 31%/27% reported good SW. Again, there were statistically significant differences in good SW between THA (p = 0.01)/TKA (p = 0.005) and the control group (Supplementary data). The proportion of women with revision arthroplasties until the end of follow-up was 21% for THA. Their results for good PC were: 92% (baseline), 77% (1st postoperative questionnaire) and 62% (2nd postoperative questionnaire). In this revised group, the proportion of women with good SW reports at the same follow-up points were 46%, 15%, and 18%. Only 6.7% of women with a TKA had revision arthroplasties.Among women with arthroplasty between baseline and 10-year follow-up, the mean age at the time of arthroplasty was 64 (THA)/65 (TKA) years. The median follow-up time for the groups was THA 13 (10–20)/TKA 12 (9–19) years. Good PC was reported by 83% of women with THA and 80% of women with TKA, at the 10-year follow-up (1st postoperative questionnaire). At the 20-year follow-up (2nd postoperative questionnaire), and good PC was reported by 64%/53% of women. The changes in good PC of women with a THA were not statistically significantly different from the control group (p = 0.2) whereas the changes in good PC in the TKA group were significantly different (p = 0.01) (Supplementary data). The changes in good PC of women with THA were not significantly different (p = 0.6) when compared with the control group. For TKA there was a statistically significant difference (p = 0.04). During follow-up checkpoints participants reported a steady decrease of SW. Eventually, at the 20-year follow-up, good SW was reported by 37% (THA) and 29% (TKA) (Supplementary data). Statistically the changes in proportion of women with good SW were significantly different from controls with THA (p = 0.004) and TKA (p < 0.001) women. Only 3.0% of the women with THA and 2.2% of women with TKA had experienced a revision arthroplasty by the end of follow-up.Among women with THA or TKA between 10-year and 20-year follow-up, the mean age at the time of arthroplasty was 70 years for both THA and TKA. The median follow-up time for these women was THA 3 (0–9)/TKA 3 (0–10) years. Good PC was reported by 76% of women with THA and 71% with TKA at the 20-year follow-up (postoperative questionnaire) , 93–95% (10-year follow-up), and 80–81% (20-year follow-up) of the propensity score matched controls. Good SW was reported by 52–61% (baseline), 52–56% (10-year follow-up), and 47–53% (20-year follow-up) .Analysis for women with THA or TKA within a 1-year period of any questionnaire showed that preoperatively 54% of THA and 65% of TKA participants reported good PC. Postoperatively good PC was reported by 62% of THA and 69% of TKA participants. Similarly, good SW was reported by 21% of THA and 24% of TKA participants preoperatively, but 37% (THA)/31% (TKA) postoperatively.Elderly women who had experienced THA or TKA maintained their self-reported PC approximately 10 years after the procedure. However, at the end of follow-up the PC and SW among women with arthroplasty generally seemed to decrease more than in women without arthroplasty. 2 prior studies reported worse physical functioning 12 years after THA than in a control group without arthroplasty  seemed to have a quite similar PC to the control group, 10 years prior to arthroplasty, at baseline. Before arthroplasty, at the 10-year follow-up, there was slight decrease in PC results, probably due to progression of osteoarthritis of the index joint. The postoperative scores in PC were close to those reported by the control group, and age-related factors may decrease patients’ physical capabilities even more than arthrosis. However, neither THA nor TKA completely restored a person’s ability to walk. The SW of these older women was good throughout the follow-up. Previous data has shown that age is not an obstacle for an effective THA or TKA and elderly people report improved quality of life scores after THA/TKA operations , include walking distance as a variable. Thus, our end point variable may be considered feasible for evaluation of the functional status. In addition, there are prior studies validating different self-reports in OSTPRE. Recently, we have reported the validity of self-reported physical capability with functional tests in the OSTPRE cohort (Juopperi et al. In conclusion, THA and TKA maintain self-reported PC and SW. Yet, the overall PC and SW are lower in women with prior arthroplasty, in comparison with age-matched controls without arthroplasty. THA seems to outperform TKA in maintaining PC.Click here for additional data file.


This study estimated thresholds for clinically important responses and minimally important differences for two indicators of improvement for the 10-item version of the functional outcomes of sleep questionnaire (FOSQ-10).Participants with excessive daytime sleepiness with narcolepsy or obstructive sleep apnea received 12 weeks of solriamfetol treatment. Participants completed the FOSQ-10 and other patient-reported outcome measures, including the single-item patient global impression of change (PGI-C) assessment. Clinicians completed the single-item clinician global impression of change (CGI-C) for each participant. Data from the two studies were analyzed separately, both without regard to treatment assignment. In total, 690 participants  were enrolled. Two clinically important changes, defined as a minimally important difference and a clinically important response, were determined using distribution and anchor-based analyses. A receiver operating characteristic analysis was used to determine the optimal FOSQ-10 change threshold.Spearman correlations between change in FOSQ-10 scores and PGI-C and CGI-C were − 0.57 and − 0.49 for participants with narcolepsy and − 0.42 and − 0.37 for participants with obstructive sleep apnea. Receiver operating characteristic analysis suggested minimally important difference and clinically important response estimates of 1.7 and 2.5 and 1.8 and 2.2 points in narcolepsy and obstructive sleep apnea, respectively.Minimally important difference and clinically important response estimates for the FOSQ-10 for adults with excessive daytime sleepiness in narcolepsy or obstructive sleep apnea will be helpful for interpreting changes over time and defining a clinical responder.NCT02348593  and NCT02348606  Excessive daytime sleepiness (EDS) can be caused by a variety of conditions, including narcolepsy, obesity, sleep apnea, and other chronic diseases . EDS is The Functional Outcomes of Sleep Questionnaire (FOSQ) is a 30-item, disease-specific, quality-of-life questionnaire designed to assess the impact of EDS on activities of everyday living and the extent to which these activities are improved by effective treatment . An abbrEstablishing thresholds that characterize a meaningful change to patients can help clinicians interpret results from patient-reported outcome measures and may inform clinical decisions regarding patient care , 12. TheWe sought to identify two thresholds for change for the FOSQ-10. The first, the minimally important difference (MID), is defined as the smallest change that a patient is able to identify. The second, the clinically important response (CIR), indicates a change that represents a relevant treatment benefit . By esta2 and examined the safety and efficacy of solriamfetol for EDS. However, some inclusion criteria differed slightly; one trial involved participants with OSA, while the other enrolled those with narcolepsy without moderate to severe OSA. Additionally, while inclusion criteria regarding the Epworth Sleepiness Scale (ESS) were the same in the two trials, there were slightly different requirements for baseline sleep latency based on the Maintenance of Wakefulness Test (MWT): patients in the OSA trial were included if their sleep latency was less than 30 min, whereas patients in the narcolepsy trial were included if their sleep latency was less than 25 min. For the OSA study, participants had OSA diagnosed according to International Classification of Sleep Disorders, 3rd edition (ICSD-3) criteria and current or prior use of a primary OSA therapy, including a CPAP machine, oral appliance, or surgical intervention [Diagnostic and Statistical Manual of Mental Disorders, 5th edition [Data were analyzed from participants with EDS associated with narcolepsy or OSA who were enrolled in one of two phase 3, multicenter, randomized, double-blind, 12-week studies , 15 of s edition  criteriaParticipants completed the MWT and ESS. The MWT is administered by trained clinicians and measures the ability to stay awake for a defined period of time, requiring participants to try to stay awake as long as possible while seated in a comfortable chair in a darkened room . The ESSData from the two studies (NCT02348593 and NCT02348606) were analyzed separately, both without regard to randomization group. The analyses were based upon the modified intent-to-treat population that includes data from participants who were randomized, received at least one dose of study medication (solriamfetol or placebo), and have baseline and at least one post-baseline evaluation.Demographic characteristics  were summarized descriptively, along with baseline values of the FOSQ-10 and ESS, using frequency and percent for categorical variables and mean, standard deviation (SD), and range for continuous variables.d or the standardized effect size [Distribution-based analyses were used to estimate the minimal detectible change (MDC), which represents the smallest change that can be reliably distinguished from random fluctuation and thus represents the lower bound for estimates of clinically important change. The distribution-based measures that served as estimates of the MDC were 1.0 standard error of measurement (SEM), a value of 0.5 Cohen’s ect size , and a vect size .Next, an anchor-based approach that incorporated either PGI-C or CGI-C (as assessed at week 12) as the anchor was used to determine what magnitude of change on the FOSQ-10 represented a meaningful change from the patient’s or clinician’s perspective. This type of approach has previously been used for interpreting changes that are meaningful on the ESS and other patient-reported outcomes , 21.Spearman correlation coefficients were calculated between each of the anchors  and the change in FOSQ-10 score from baseline to week 12 or early termination. Correlations ≥ 0.30 in absolute value are recommended to demonstrate suitable anchors and to avoid contamination of interpretation thresholds with noise . ParticiWe evaluated two thresholds for determining a CIR. The first was a PGI-C or CGI-C rating of “minimally improved” or better, and the second was a rating of “much improved” or better. To characterize the association between each specific FOSQ-10 change score and this definition of a CIR, the sensitivity and specificity were calculated, and receiver operating characteristic (ROC) curves were derived using logistic regression analyses. ROC curves simultaneously describe the sensitivity and specificity of a predictive measure as different cutoff values are applied. The optimal values for the FOSQ-10 changes that were best associated with each of the PGI-C or CGI-C threshold values for clinically important change  were generated from the corresponding ROC curves. The area under the ROC curve, reported as the C-statistic from the logistic regression model, represents the overall ability of model predictions to discriminate between individuals who do and do not experience clinically important change at the specified level.N = 231), 65% were female, 80% were white, and 81% were from North America, and the mean age was 36 years. The mean (SD) ESS score for these participants was 17.2 (3.18), and the mean (SD) FOSQ-10 score was 11.7 (3.03). Among participants with OSA (N = 459), 38% were female, 76% were white, and 97% were from North America, and the mean age was 54 years. OSA participants had a mean (SD) ESS score of 15.2 (3.32) and a mean (SD) FOSQ-10 score of 13.9 (3.01).The analysis included a total of 690 participants across the two studies Table . Among pd, and 0.5 Guyatt’s statistic were 1.26, 1.49, and 1.03, respectively. In participants with OSA, the values were 1.48, 1.51, and 1.21, respectively.In participants with narcolepsy, the 1.0 SEM, 0.5 Cohen’s Spearman correlations between each of the anchors, PGI-C and CGI-C, and changes in FOSQ-10 from baseline to week 12 or early termination were − 0.57 and − 0.49 for participants with narcolepsy and − 0.42 and − 0.37 for participants with OSA, confirming that both ratings were suitable for use in anchor-based MID analysis . In geneThe mean changes in FOSQ-10 scores for those who rated themselves (PGI-C) or whose clinician rated them (CGI-C) as “minimally improved” were 2.05 and 1.74 for narcolepsy and 2.08 and 1.66 for OSA. These values exceed the highest estimated value for the MDC, approximately 1.5 in both indications. In participants who were rated as “much improved” on the PGI-C or CGI-C, the mean changes in FOSQ-10 were 4.03 and 4.28, respectively, for participants with narcolepsy and 3.32 and 3.40 for participants with OSA  are presented in Table The C-statistics (areas under the ROC curve) for the analyses of FOSQ-10 changes associated with PGI-C or CGI-C ratings of “much improved” or “very much improved” were between 0.71 and 0.79, indicating good accuracy for predicting a true response Fig. . For refIn the analyses of FOSQ-10 changes associated with PGI-C or CGI-C ratings of “minimally improved,” “much improved,” or “very much improved,” the C-statistics were between 0.67 and 0.76 Fig. . SensitiThis aim of this study was to provide guidance on how to interpret within-patient changes in the FOSQ-10 over time by estimating thresholds for the MID and CIR. Multiple approaches were employed to provide an estimate of two levels of patient-perceived change in the FOSQ-10 total score: that of minimal change (MID) and the CIR. This work provides clinicians and researchers with benchmarks for evaluating and interpreting FOSQ-10 results in a manner that associates the patient or clinician perception of improvement with the FOSQ-10 total score. To ensure a robust estimate, we used multiple anchors and integrated results from both anchor-based and distribution-based estimates, as well as ROC analyses, in accordance with established recommendations .This study also provided the opportunity to compare levels of change in the FOSQ-10 in participants with narcolepsy and OSA. The MID values for narcolepsy and OSA participants were 1.7 and 1.8, respectively, and the CIR values for narcolepsy and OSA participants were 2.5 and 2.2, respectively.There is increasing recognition that the clinical significance of a change cannot be inferred from statistical significance alone. Determining clinical significance requires an understanding of how the change is perceived and experienced by the patient. The estimation of the MID and CIR provides clinicians and researchers with benchmarks for two levels of change, as perceived by patients, specifically the smallest change that a patient can identify (MID) and that which represents a relevant treatment benefit (CIR). Having both benchmarks available provides the opportunity to evaluate the levels of change of FOSQ-10 scores that may occur over time. The use of many of the anchor-based and distribution-based methods employed in the current study traces back to the early 1990s , and theThese analyses have several notable strengths. First, the analyses incorporated both distribution- and anchor-based methods. Two anchors, one based on a PGI-C and the other on a CGI-C, were used. Notably, the anchors reflected similar mean changes in FOSQ-10 scores, thereby strengthening the interpretation of what changes in FOSQ-10 scores represent. Additionally, the estimation of the MID and CIR was derived in two distinct patient populations with EDS. However, a limitation is that few participants in the current study experienced a worsening in their condition, precluding the estimation of either an MID or a CIR for worsening symptoms. This is consistent with results previously reported from these studies that indicated improvements in EDS with solriamfetol treatment, as demonstrated by least square mean decreases in ESS scores  and increases in mean MWT sleep latency  from baseline to week 12 relative to placebo  , 15. ThiDespite these limitations, the CIR estimate for the FOSQ-10 overall score of 2.2 to 2.5 for adult populations with OSA or narcolepsy treated for EDS will be valuable for interpreting changes over time and defining a clinical responder. Future research in other populations or those receiving other therapies for EDS will add to this work.


We and others have previously demonstrated that the endothelium is a primary target of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and L-arginine has been shown to improve endothelial dysfunction. However, the effects of L-arginine have never been evaluated in coronavirus disease 2019 (COVID-19).This is a parallel-group, double-blind, randomized, placebo-controlled trial conducted on patients hospitalized for severe COVID-19. Patients received 1.66 g L-arginine twice a day or placebo, administered orally. The primary efficacy endpoint was a reduction in respiratory support assessed 10 and 20 days after randomization. Secondary outcomes were the length of in-hospital stay, the time to normalization of lymphocyte number, and the time to obtain a negative real-time reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2 on nasopharyngeal swab. This clinical trial had been registered at ClinicalTrials.gov, identifier: NCT04637906.p < 0.01) had the respiratory support reduced; however, a significant difference was not detected 20 days after randomization. Strikingly, patients treated with L-arginine exhibited a significantly reduced in-hospital stay vs placebo, with a median  of 46 days  in the placebo group vs 25 days  in the L-arginine group (p < 0.0001); these findings were also confirmed after adjusting for potential confounders including age, duration of symptoms, comorbidities, D-dimer, as well as antiviral and anticoagulant treatments. The other secondary outcomes were not significantly different between groups.We present here the results of the initial interim analysis on the first 101 patients. No treatment-emergent serious adverse events were attributable to L-arginine. At 10-day evaluation, 71.1% of patients in the L-arginine arm and 44.4% in the placebo arm (In this interim analysis, adding oral L-arginine to standard therapy in patients with severe COVID-19 significantly decreases the length of hospitalization and reduces the respiratory support at 10 but not at 20 days after starting the treatment.Both placebo and L-arginine were kindly provided by Farmaceutici Damor S.p.A., Naples The severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) infection has spread worldwide, causing disease and mortality, as well as social disruption and economic loss. Endothelial cells have been shown to be a cardinal target in COVID-19, and L-arginine is known to improve endothelial dysfunction. PubMed and medRxiv preprint searches were updated on June 24, 2021 using the search criteria "SARS-CoV-2" "COVID-19" “L-arginine”. These searches retrieved no items.vs placebo (log-rank p <  0.0001). Furthermore, at the 10-day evaluation, 71.1% of patients in the L-arginine group and 44.4% in the placebo group (p < 0.01) had reduced the respiratory support; however, a significant difference was not detected 20 days after randomization, most likely because most of the participants in the L-arginine arm had been already discharged from the hospital by this time.The first interim analysis of this randomized, double-blind, placebo-controlled, parallel-group trial shows that patients hospitalized for severe COVID-19 treated with L-arginine displayed a significantly reduced in-hospital stay There is concrete evidence that endothelial dysfunction is a fundamental feature of COVID-19. However, no clinical trial has actually shown that intervention improving endothelial function could be beneficial in COVID-19. Our interim findings indicate for the first time that adding L-arginine orally to standard therapy in patients with severe COVID-19 significantly reduces the length of hospital stay and respiratory support.Alt-text: Unlabelled box1The clinical course of coronavirus disease 2019 (COVID-19) consists of two main phases: viral infection and immune/inflammatory response, requiring distinct therapeutic approaches to stop virus replication and to attenuate the inflammatory state that is commonly observed in COVID-19 patients and may contribute to multiorgan failure Clinical and preclinical evidence supports the view that the endothelium is a key target organ in COVID-19, providing a mechanistic rationale behind its systemic manifestations On these grounds, given the well-established beneficial effects of L-arginine on endothelial function 22.1Domenico Cotugno Hospital . The study protocol is reported in Appendix 1.We designed an investigator-initiated, individually randomized, placebo-controlled, double-blind trial aiming to assess the effectiveness and safety of oral L-arginine in adults (aged ≥ 18 years) admitted to hospital with severe COVID-19, defined as previously reported by other investigators ,20. The Ethical approval was obtained from the institutional Ethical Committee of the Hospital. Written informed consent was collected from all patients, or their legal representative if they were unable to provide consent. The trial was performed in accordance with the principles of the Declaration of Helsinki and the International Conference on Harmonization-Good Clinical Practice guidelines.Eligible hospitalized patients were men and non-pregnant women with COVID-19 who were at least 18-year-old and were RT-PCR positive for SARS-CoV-2; patients were screened by applying the following eligibility criteria.2.2•Pneumonia confirmed by chest imaging;•Oxygen saturation of 93% or lower on room air;•2/FiO2, or P/F) of 300 or less;Ratio of alveolar oxygen partial pressure to fractional inspired oxygen  was taken into account during randomization. Protocol adherence was assessed daily during the hospitalization period. Patients, ward doctors, trial personnel and outcome assessors were unaware of the type of supplementation provided. All clinical interventions, including the use of antibiotics, corticosteroids, anticoagulants, ventilation strategy, and laboratory investigations were at the discretion of the treating physicians according to clinical needs for both treatment groups.Eligible patients were assigned, with a 1:1 ratio, based on a computer-generated randomization table, to add to the hospital standard therapy 1 bottle containing 1.66 g of L-arginine  or 1 bottle of identical aspect not containing L-arginine, twice a day orally for the whole hospitalization period. The formulation of Bioarginina® used in this study consists of oral vials containing 1.66 g of L-arginine, whose taste is fully buffered by the presence of sucrose and by an acidity regulator (anhydrous citric acid); therefore, there were no differences in appearance, smell, or taste between treatment and placebo. The dose was chosen based on a previous study proving that such a regimen was safe and significantly effective in ameliorating the oxidative metabolism of professional water polo players 2.42 from arterial blood gas test; FiO2 was considered 0.21 when the patient was breathing room air A single follow-up form was completed by the trial staff when each patient was discharged or had died, whichever occurred first. Information was recorded regarding the patients’ adherence to the assigned treatment, administration of other treatments, duration of symptoms on admission, duration and type of respiratory support, renal dialysis or hemofiltration. In addition, we obtained routine health care and registry data, including information on vital status (or date and cause of death), discharge from the hospital, as well as respiratory and renal support therapy. The P/F ratio was calculated as previously described ,24, obtaBefore randomization, the demographic characteristics and other clinical and laboratory data required by the routine diagnostic activity were collected. These parameters were evaluated daily during the hospitalization period according to routine hospital practice. The safety assessment included daily monitoring for adverse events, clinical laboratory testing, 12-lead electrocardiogram, and daily measurement of vital signs.2.5The primary clinical endpoint was a reduction in respiratory support , evaluated 10 and 20 days after randomization.Secondary outcomes were the length of in-hospital stay, the time to lymphocyte number normalization, and the time to obtain a negative RT-PCR for SARS-CoV-2 on nasopharyngeal swab.Safety outcomes included treatment-emergent adverse events, serious adverse events, and premature discontinuations of study drug.2.6Since no reliable data were available for an accurate calculation of the sample size, we harnessed data from our preliminary clinical experiences conducted with the administration of L-arginine in COVID-19 patients and those of a historical group of COVID-19 patients undergoing the same therapeutic regimen except the daily oral supplementation of L-arginine. Based on these data, we hypothesized a difference of at least 35% in the primary outcome between the two study groups. According to this hypothesis, we determined that it was necessary to enroll a population of 290 patients to detect a statistically significant difference with a 2-side α level of 0.05 and a power of 80%.p < 0.01 for efficacy and p < 0.01 for tolerability. Enrollment was planned to continue until the Data and Safety Monitoring Board (DSMB) recommended stopping the trial for evidence of efficacy, futility, or harm, based on evaluation of all the available data, including data internal and external to the trial.Two interim analyses were planned for the assessment of efficacy and tolerability after completion of the first 100 and 200 patients. The study discontinuation threshold was set at th percentile, 75th percentile) for severely skewed continuous variables and as numerical values (percentages) for categorical variables. Normal distribution of the data was verified via normal probability plots and confirmed with the skewness/kurtosis test for normality. Bivariate tests were used to assess the association between the treatment with L-arginine and the baseline characteristics and treatments of the patients included in the study. Statistical significance was determined by a p value < 0.05. In the statistical analysis, differences for continuous variables were evaluated using two-sample t-test for approximately normally distributed variables and Mann-Whitney U test for severely skewed variables. Chi-square or Fisher tests were used to measure associations between dichotomous and categorical variables.The characteristics of the participants are reported as the mean ± standard deviation (SD) for approximately normally distributed continuous variables, as the median , adjusting for potential confounders; a backward selection method was applied to create the final model. Log-rank test was used to compare the length of in-hospital stay between the L-arginine and the placebo groups and a Cox proportional hazards regression analysis was employed to relate L-arginine treatment to length of hospitalization adjusting for likely confounders. All analyses were performed using SPSS 26.0.2.7Both placebo and L-arginine were kindly provided by Farmaceutici Damor S.p.A., Naples, Italy, which had no role in the design and conduct of the study, collection, management, analysis, and interpretation of the data, preparation, review, or approval of the manuscript, and decision to submit the manuscript for publication. GF, RI, and BT had full access to all data in the study and take final responsibility for manuscript submission.3Between November 2020 and April 2021, 210 patients were screened, of whom 101 were eligible ; 48 patip < 0.01) had reduced the respiratory support  assessed 10 and 20 days after randomization. At 10-day evaluation, 44.4% in the placebo group and 71.1% in the L-arginine group .A multivariable logistic regression was performed in order to examine the magnitude of association between the treatment with L-arginine and the primary outcome and to better identify explanatory variables that were associated with a reduction of the respiratory support . When advs L-arginine: 228.3 ± 93; p = 0.02) but not at 20 days (Placebo: 231.0 ± 93 vs L-arginine: 263.5 ± 103).On the contrary, 20 days after randomization we failed to detect any significant difference in the primary outcome between the study arms . Similarly, the P/F ratio was significantly different at 10 days . This association remained significant in a Cox regression analysis . The variables used in the regression analyses were selected according to the available literature on COVID-197-9,11.The multivariable logistic regression performed on the ITT population at 10 and 20 days after randomization is presented as supplementary material . Among the secondary outcome measures, differences between active treatment group nine arm . Specifianalysis  using thp = 0.21).The rate of lymphocyte number normalization was similar between the 2 study arms . The time to obtain a negative RT-PCR for SARS-CoV-2 on nasopharyngeal swab was comparable between groups  deceased shortly after the transfer, before they could actually start the treatment with L-arginine or placebo. Since these patients were transferred to the ICU right after the randomization, they have been included in the ITT but not in the PP analysis. Besides, 3 other patients in the placebo arm (0 in the L-arginine arm) died after day 20. Thus, the total mortality was 3.3% in the PP analysis  and 13.9% in the ITT analysis .3.1Four serious adverse events were reported  which were considered unrelated to the study treatment by the investigators.4In this randomized clinical trial involving adults with severe COVID-19, L-arginine plus standard care compared with standard care alone significantly reduced the need for respiratory support and, foremost, the duration of hospitalization. L-arginine was not associated with increased risk of adverse events in these critically ill COVID-19 patients. Notably, the safety of oral L-arginine had been previously ascertained in other trials ,27.th,75th percentile) number of days was 46  in the placebo group vs 25  in the L-arginine group ; this finding was also confirmed after adjustment for potential confounders including age.We selected the reduction of respiratory support as the primary endpoint because it comprises both pathophysiologic and clinical determinants. Thus, in order to assess the role of L-arginine supplementation in the reduction of respiratory support, we performed a multivariate analysis which demonstrated that treatment with L-arginine was the main determinant of this phenomenon, ruling out the possibility that it could be merely due to the younger age of this group of patients. This conclusion is corroborated by the observation that L-arginine supplementation was the main determinant in the reduction of in-hospital stay duration; the in-hospital stay median , IL-10, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) in COVID-19 patients [T lymphocyte apoptosis by activating extrinsic and intrinsic apoptosis pathways during SARS-CoV-2 infection [T cell function, which has been shown to depend on L-arginine supply In vitro assays revealed that T cell proliferative capacity is significantly reduced among COVID-19 patients and can be restored through L-arginine supplementation T cell function may be of greater importance than the mere impact on lymphocyte number.The exact mechanisms linking SARS-CoV-2 infection and lymphopenia remain not fully defined patients ,47. Highnfection ,49. Whatnfection ,42,50. TIntriguingly, diabetes was significantly associated with the primary outcome but not with the hospitalization length; this observation could be related to the previously reported detrimental role of diabetes on lung function ,57; the At day 20 after randomization, we did not detect any significant difference in the rate of patients that transitioned to a lower respiratory support; however a nearly twice the number of participants in the L-arginine arm had been already discharged from the hospital by this time, leaving on a small number of patients remaining-likely the sickest patients of the cohort; therefore, we can speculate that many of the patients with earlier discharge would have demonstrated improvements had they been analyzed at day 20.The main limitation of our study is the relatively small number of patients included in this first interim analysis; therefore, further studies in larger populations are warranted, as also suggested by the high confidence interval (CI) around L-arginine in Strengths of this trial included its blinded, placebo-controlled design, high adherence to the study protocol, rigorous monitoring for safety and adverse events, and rapid recruitment. Moreover, all patients were analyzed according to their randomization group, and follow-up was completed. We included patients with respiratory symptoms for up to 15 days prior to randomization while some trials of antiviral medications limit enrollment to patients with symptoms for a shorter duration, most likely in an effort to enrich the population for patients most likely to benefit.T cell impairment are consequences of low L-arginine bioavailability and contribute to complications of COVID-19, making a role for L-arginine supplementation biologically plausible. Thus, our results may have important clinical implications for COVID-19 treatment especially in low-resource environments were vaccination is not widely available given the safety and the affordability of oral L-arginine.Finally, it should be noted that both endothelial dysfunction and Taken together, the results of this interim analysis indicate that adding L-arginine to standard therapy in patients with severe COVID-19 significantly decreases the length of hospitalization and reduces the respiratory support assessed 10, but not 20, days after starting the treatment. The other secondary outcomes were not significantly different between groups.We declare no competing interests.


Seven patients with peri-implant disease applied dual-light aPDT daily in addition to their normal dental hygiene for four weeks. A single Lumoral® treatment includes an indocyanine green mouth rinse followed by 40 J/cm2 radiant exposure to a combination of 810 nm and 405 nm light. A point-of-care analysis of active-matrix metalloproteinase (aMMP-8), visible plaque index (VPI), bleeding on probing (BOP), and peri-implant pocket depth (PPD) measurements was performed on day 0, day 15, and day 30. Reductions in aMMP-8 (p = 0.047), VPI (p = 0.03), and BOP (p = 0.03) were observed, and PPD was measured as being 1 mm lower in the implant (p = ns). These results suggest a benefit of regular application of dual-light aPDT in peri-implantitis. Frequently repeated application can be a promising approach to diminishing the microbial burden and to lowering the tissue destructive proteolytic and inflammatory load around dental implants. Further studies in larger populations are warranted to show the long-term benefits.Until now, in clinical dentistry, antibacterial photodynamic therapy (aPDT) has been restricted to in-office treatments, which hampers repeated applications. This pilot study tested the benefit of a commercially available Lumoral Dental implants have been used to replace lost teeth for decades . Dental Antibacterial photodynamic therapy (aPDT) and antibacterial blue light (aBL) are emerging treatment methods auxiliary to mechanical debridement for periodontitis and peri-implantitis ,11,12,13®/ImplantSafe®/ORALyzer®) are commercially available as quantitative point-of-care kits for periodontitis and dental peri-implantitis. Accurate quantitative reading of aMMP-8 can be conveniently performed chairside [Collagenase-2/neutrophil collagenase or matrix metalloproteinase (MMP)-8 is a collagenolytic host proteinase. The active form (aMMP-8) reflects and predicts periodontal tissue destruction and inflammation. The aMMP-8 is a promising candidate biomarker used to diagnose, grade, and predict periodontitis and peri-implantitis as well as to monitor their treatments ,27,28,29hairside ,28,30,31hairside .Repeated antibacterial treatment by dual-light aPDT performed at home can be used to improve oral hygiene. The aim of the study was to preliminarily test the ability of the anti-plaque and anti-inflammatory effects of the daily application of dual-light aPDT in peri-implant disease patients, monitored by the aMMP-8 point-of-care method. We hypothesized that improved oral hygiene performance by dual-light aPDT would reduce collagenolytic inflammation and burden without adverse effects.The study was conducted in accordance with the ethical principles of the Declaration of Helsinki. The study was approved by the ethics committee of the Hospital District of Helsinki and Uusimaa (HUS/1271/2019), and all participants provided written informed consent before enrolment.®. The patients were asked to continue with their regular home care and routine oral hygiene habits during the study period. The patients were instructed to perform an improved oral hygiene protocol by including dual-light aPDT in their regular home care for four weeks before the actual mechanical maintenance treatment. The patient characteristics are presented in This experimental pilot study followed an interventional pre-post protocol. The inclusion criteria were (1) implant patients arriving for a maintenance visit at a dental clinic ; (2) a diagnosis of peri-implant mucositis or peri-implantitis by a dentist according to the definitions described below; and (3) written informed consent. The exclusion criteria included (1) smoking; (2) diabetes; (3) a need for a hopeless teeth extraction, or open cavities in need of immediate endodontic treatment; or (4) the presence of a major physical limitation or restriction that prohibits the hygiene procedures used in the study protocol. Seven patients were enrolled, four males and three females. Their ages ranged from 65 to 89 years. A single implant with screw crown in each patient was selected for the study, with three implants being from the mandibulae and five being from the maxilla. All implants were produced by Nobel Biocare® 8 mobile phone 12 MP camera with f/1.8 aperture, six-element lens, optical stabilization, and Wide Color capture deployment .The dental examinations were performed at the beginning of the study (time point 1), after 15 days (time point 2), and after 30 days (time point 3). An oral clinical investigation at each time point included an assessment of the visible plaque index (VPI) , BOP , and PPD (mm). A millimeter-grade Ball-Tip Screening probe, WHO, AEEP23/WHOBX  was used. A digital intra-oral radiograph was acquired using Sirona Heliodent plus , and Soredex Digora Optime  software was used for the analysis of alveolar bone destruction to define peri-implantitis ,34. Clinn = 3) was based on the presence of peri-implant signs of inflammation described above in combination with a lack of additional bone loss following initial healing. Peri-implantitis (n = 4) was defined by the presence of peri-implant signs of inflammation with radiographic evidence of bone loss (>2 mm) and increased probing depths ≥ 3 mm with the presence of BOP, swelling, and possible suppuration [The diagnostic definition of peri-implant health and disease was based on the criteria published by Renvert et al.  and Bergpuration ,34.® mouthwash  in conjunction with a Lumoral® light applicator . Lumorinse®  is a CE-marked effervescent tablet including 7 mg of ICG and is used by dissolving in 30 mL of water, which produces a mouthwash with an ICG concentration of 250 µg/mL. The mouthwash is swished for 1 min, enabling ICG to adhere to dental plaque before light application. Lumoral®  is a CE-marked medical device providing simultaneous 405 nm aBL and 810 nm near-infrared (NIR) LED light in a mouthguard form. Together with Lumorinse® , the product offers simultaneous aBL and aPDT action on dental plaque. The Lumoral® light applicator is placed in the mouth, and during 10 min use, the device delivers a radiant exposure of 40 J/cm2 light with approximately half of the light energy as aBL and half as NIR light. Thus, the device gives light to the whole dentition, with the 48 LEDs illuminating both dental arches  and ORALyzer® digital aMMP-8 reader  were used to quantify aMMP-8 (ng/mL) in PISF. The PISF samples were obtained by inserting a collection strip into the peri-implant pocket in the sulcus point exhibiting the worst clinical situation for 30 s. Then, according to the manufacturer’s instructions, the strip was place in the elution fluid for five minutes. After five minutes of incubation, the elution tube was swirled a couple of times. The dipstick was then immersed into the elution fluid for 15 s and placed into the reading compartment of the ORALyzer® device . The ImplantSafe® test  can read qualitatively after 5 min; the reading window shows one blue line  or two lines . The ORALyzer® device  read the test quantitatively. The aMMP-8 enzyme test results were documented by photography (iPhone 8).The ImplantSafep-value less than 0.05 was considered statistically significant.SAS 9.4 software was used for the power calculations  and GraphPad software version 9.1.0  to analyze the data and to create the graphs. A Wilcoxon nonparametric analysis of paired groups was performed to compare the difference of continuous variables  and chi-square contingency analysis for the dichotomous variable (BOP) between the pretreatment and final measurements. A We performed a power calculation for the required sample size based on our recently published study on ICG-assisted aPDT on dental plaque . In thisCalculated  from the previous study design and assigning 5% for alpha errors and 20% for TYPE II errors (80% power), we arrived at a sample size of six patients . To alloAll seven participants, aged 65–89 years, completed the whole protocol. None of the participants had diabetes or rheumatoid arthritis, and they were all non-smokers. One had asthma, and two had a diagnosis of heart disease. The year of placement of the prosthetic reconstruction ranged from 2009 to 2016, with a median of 2.62 years since placement.At the beginning of the study (time point 1), all study subjects had gingival bleeding at the area of the selected implant (BOP 7/0). The amount of dental plaque by VPI ranged from 0 to 2, with a median of 1. The probing depth ranged from 2 to 6 mm with a median of 4 mm. The aMMP-8 ranged from 10 to 170.8 ng/mL, with a 72.6 ng/mL median. In four implants, X-ray imaging showed >2 mm peri-implant bone loss from the estimated bone level.At time point 2, after two weeks of once-a-day dual-light aPDT use, two subjects showed negative BOP at the area of the selected implant (BOP 5/2). One patient showed a reduction in plaque, with VPI reducing from 1 to 0. The others had no change in the amount of dental plaque. The probing depth stayed the same in all subjects. The aMMP-8 level measurements ranged from 10 to 138.9 ng/mL, with a 37.1 ng/mL median.At time point 3, after another two weeks of regular dual-light aPDT use but now twice a day, three subjects had no bleeding at the gingiva around the studied implant (BOP 4/3). VPI showed a level-one plaque score in one patient, while all others had scores of 0. One patient had a reduction of 1 mm in probing depth, while others showed no change. The aMMP-8 level measurements ranged from 10 to 109.8 ng/mL, with a median of 23.6 ng/mL . No adveThis initial study suggests a benefit of home-applied dual-light aPDT in patients with peri-implant disease. We observed improved hygiene in all subjects measured by a reduction in VPI. The aMMP-8 in PISF levels were clearly reduced, being in line with the decrease in inflammation measured by BOP. In one implant, a 1 mm reduction in PPD was measured four weeks after the beginning of the study. The significance of this observed reduction in inflammation lies in none of the study subjects receiving any professional treatment until after the study. All improvements were achieved at home by enhanced oral hygiene.The root cause of peri-implant disease is tissue reaction to bacterial biofilm. Mechanical biofilm removal is the standard of care for disease management and aims for a reduction in bacterial colonization on the surface of the implant ,36. BecaStrong evidence supports the use of aMMP-8 in oral fluids in the assessment of the activity and severity of peri-implant disease . Peri-imWe observed a negative BOP in three studies at four weeks after dual-light aPDT use. The absence of BOP is a precise measurement for periodontal stability with a negative predictive value of 98% . This isAntibacterial photodynamic therapy and aBL have been presented as new antibacterial methods. Both methods provide a significant dose-dependent antibacterial action ,16. Nikiwww.lumoral.com; Koite Health LTD, Espoo, Finland). LED technology can bring the treatment location from clinics to home. Antibacterial photodynamic therapy is a potent antibacterial tool, but results in periodontitis have been generally disappointing. In most of these studies, the treatment has been applied only once [The rapid development in LED technology has provided an opportunity to attack dental plaque  with lignly once . In the nly once ,46. Treanly once . We clainly once . In the nly once . Indeed,® , where the use of a placebo is particularly difficult [This is a small study with a limited population size and a relatively short study period of only one month. Our power calculations, based on a restricted number of available patient data, are weak and provide an initial reference at best. With an increasing number of patients being treated in small but cumulating pilot experiments, it is our aim to obtain further evidence for designing larger clinical trials with enough data to perform power calculations with better resolution and higher confidence. However, the observations reported here definitely encourage further studies. The use of a cross-over design would be a good option when studying a treatment such as Lumoralifficult . Use of ifficult ,50,51. Cifficult .This pilot study suggests that dual-light therapy might be a promising adjunctive homecare tool to keep high-cost implants secure from peri-implantitis. It is easy and safe to use without any adverse effects. However, further studies are warranted to define the effectiveness of the treatment.